Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Non-invasive in vivo imaging requires near-infrared (NIR) fluorescent probes. Recent development of genetically encoded fluorescent proteins (FPs) from bacterial phytochrome photoreceptors (BphPs) has significantly advanced deep-tissue and whole-body imaging (1). In contrast to far-red GFP-like FPs, BphP-derived FPs are excited and fluoresce close to or within an NIR tissue transparency “optical window” (˜650-900 nm) where background autofluorescence is low, light scattering is reduced, and combined absorption of hemoglobin, melanin, and water is minimal (2).
NIR fluorescence of BphP-based FPs results from an incorporation of the most red-shifted natural chromophore, biliverdin IXα (hereafter BV) (1, 3, 4), that is similar to their parental BphPs (5, 6). Fortunately, BV is abundant in eukaryotes, including mammals, as an intermediate of heme degradation pathway to bilirubin (7, 8). In wild-type BphPs, light absorption results in BV isomerization and conformational changes of the protein backbone, leading to activation of an output effector domain. In engineered NIR FPs, the photoisomerization is blocked and the other non-radiative energy dissipation pathways are suppressed by truncation of BphPs to the chromophore-binding PAS-GAF domains and by introducing of amino acid substitutions in the chromophore immediate environment (1, 9).
Although BphP-based NIR FPs are now widely used in many areas of basic and translational research, including cancer studies, stem cell biology, neuroscience, and parasitology, these FPs are mainly serve as passive whole-cell labels for non-invasive in vivo imaging (5). So far these NIR FPs had the limited use in monitoring of active cellular processes in animals, such as activation of signaling cascades and protein-protein interactions (PPIs). A development of active MR reporters and biosensors, which respond to cellular events and consequently change their fluorescence, has been hampered by a lack of bright monomeric NIR FPs as building blocks for these sensors. The monomeric NIR FPs are also required to label (tag) intracellular proteins. Currently available monomeric far-red GFP-like FPs, including mKate2 (10), TagRFP657 (11), mCardinal and mNeptune2.5 (12), are suboptimal for deep-tissue imaging because their excitation maxima do not exceed 611 nm.
Current BphP-based NIR FPs have limitations and cannot be used to label proteins and to build NIR biosensors. There are three characteristics of NIR FPs, which are crucial to consider for their applications (1). The first one is an effective brightness of NIR FP in mammalian cells, which depends on its molecular brightness, intracellular stability, efficiency of BV incorporation, and cell expression level. In contrast to GFP-like FPs, the effective brightness of BphP-based NIR FPs does not always correlate with their molecular brightness (1). Decreased cellular fluorescence of some NIR FPs results from a low specificity of BV binding and a competition between BV and other heme-derived compounds, including protoporphyrin IX, for binding to NIR FP apoproteins (13, 14). The second characteristic to consider is an oligomeric state of FPs. Only monomeric FPs can be used in protein fusions without interference with functionality of the tagged protein partner (15). The third characteristic is the spectral properties of NIR FPs. Spectrally distinct NIR FPs are required for biosensors and for multicolor NIR labeling.
Among the reported BphP-based FPs, five spectrally distinct NIR FPs, iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720 (1, 4, 16) fully rely on endogenous BV and do not require its external supply or co-expression of heme oxygenase (HO). Therefore, these proteins can be used as easy as GFP-like FP by delivering a single gene to cells. Importantly, possible endogenous BV concentration variability does not influence performance of iRFPs. Indeed, iRFP713 fluorescence was observed in all tissues of two iRFP713-transgenic mouse lines (8). In both mouse lines, the iRFP713 fluorescence intensity was generally uniform in almost all organs and tissues, with slightly higher expression levels in liver, lungs, and pancreas. However, iRFPs are dimers and can mainly serve for labeling of organelles and whole cells.
The first monomeric BphP-based FP, IFP1.4 (3), is dim and do not fluoresce without a BV supply. Moreover, it forms dimers, as was found recently (17). Its brighter version IFP2.0(18) was also found to be dimeric (1, 17). Previously reported monomeric FPs, Wi-Phy (9) and IFP1.4rev (19), were characterized only in vitro (9, 19). Recently reported monomeric mIFP (17), which is the only one monomeric FP tested in cellular fusions, is dimmer than dimeric iRFPs and requires a supply of BV via co-expression of BV-producing enzyme, HO. Also, a lack of spectrally distinct versions of monomeric BphP-based FPs prevents two-color NIR protein labeling and a development of NIR reporters and biosensors.
Previously reported methods of NIR FP monomerization (3, 9, 18) resulted in significant loss of brightness in mammalian cells or were not efficient enough to prevent dimer formation at concentrations above 10 μM (more than 0.35 mg/ml for a typical BphP-based FP) (1, 17)
Thus, there is a need in the art for the development of bright monomeric spectrally distinct NIR FPs that find use in scientific applications without technical limitations due to oligomerization. There exists also a need for methods to produce such FPs.
Here we report a set of three bright spectrally distinct monomeric NIR FPs, called miRFPs, which fully rely on endogenous BV to fluoresce in mammalian cells and mammals. We demonstrate a use of miRFPs in a wide range of NIR protein tags, reporters and biosensors. First, we created a set of miRFP protein fusions and showed that they can be imaged using common diffraction-limited and super-resolution microscopy. Second, using miRFPs as scaffolds, we developed spectrally distinct monomeric bimolecular fluorescence complementation (BiFC) reporters for PPIs and for low-background RNA imaging. Third, we demonstrated a use of miRFPs to develop NIR reporters for signaling cascades and cell fate. Specifically, we designed NIR IκBα and NIR cell cycle reporters and showed that they perform well in applications across scales: from microscopy and flow cytometry to whole-body imaging.
Here we also report a method, which we applied to monomerize existing dimeric NIR FPs, termed iRFPs, without significant decrease in brightness in mammalian cells. The monomerized versions of these iRFPs were also named as miRFPs with the numbers corresponding to the emission maximum. The method can also be applied to monomerize other NIR FPs derived from BphPs.
The present invention satisfies the needs stated above and provides additional advantages. The present invention addresses the need for bright spectrally distinct genetically encoded monomeric near-infrared FPs, uses thereof, and methods to produce these FPs.
The present invention also provides NIR fluorescent reporters based on the engineered monomeric NIR FPs and uses thereof.